Detection of H. Pylori Utilizing Unlabeled Urea

ABSTRACT

Disclosed is a method and device for detection of  H. Pylori  in breath emissions utilizing an unlabelled urea, in which a patient ingests a safe quantity of unlabelled urea. After ingestion, expired breath of the patient is analyzed for ammonia, with a detection based on levels of ammonia lower than 50 ppb to 500 ppm to detect  helicobacter pylori.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation application of U.S. application Ser. No. 12/678,654, filed Mar. 17, 2010, which is a 371 of International application PCT/US08/076,631 filed Sep. 17, 2008, which claims benefit of priority of Provisional Application Ser. No. 60/973,066 filed Sep. 17, 2007, all of which are incorporated herein by reference.

BACKGROUND

1. Field of the Invention

The present invention relates generally to a medical device and protocols to facilitate diagnosis of Helicobacter pylori (H. pylori) based on administration of unlabelled urea.

2. Background of the Invention

Exhaled breath has long been known to enable non-invasive disease detection. Exhaled gases, such as ammonia, nitric oxide, aldehydes and ketones have been associated with kidney and liver malfunction, asthma, diabetes, cancer, and ulcers. Other exhaled compounds like ethane, butane, pentane, and carbon disulfide have been connected to abnormal neurological conditions. However, though analysis of body fluids (blood, sputum, urine) for disease diagnoses and monitoring is routine clinical practice, human breath analysis methodologies that exploit the non-invasive nature of such diagnoses are still under-developed and conventional technologies lack specificity, are excessively expensive or lack portability.

Sensors have been produced to measure gases in a variety of settings, including automotive and biological applications. See U.S. Pat. No. 7,017,389 to Gouma, the contents of which are incorporated by reference, regarding detection of No_(x) emissions in the automotive field. Technologies for monitoring exhaled breath require complex and expensive apparatuses that are difficult to calibrate and are often not sufficiently sensitive to provide a high degree of certainty in regard to medical condition diagnosis. Such biological systems pose challenges that include sensitivity to extremely low levels of gases, presence of reducing and oxidizing gases, organic vapors (VOCs), etc. See U.S. Pat. No. 7,220,387 to Flaherty et al., the contents of which are incorporated by reference, regarding disposable sensors to measure gaseous sample analytes.

A conventional apparatus disclosed by Kearney, D, et al., Breath Ammonia Measurement in Helicobacter pylori Infection, Digestive Diseases and Sciences, Vol. 47, No. 11, pp. 2523-2530 (2002), provides a fiber optic device placed in the stream of expelled breath that is connected to an optical sensor for detecting whether a patient has H. pylori by measuring for ammonia excreted by the lungs, utilizing a hydrophobic TFE-based membrane to avoid affect of dissolved ions such as ammonia. Also see, WO 03/041565 A2 of Hubbard et al., the contents of which are incorporated by reference, H. pylori detection.

Diagnostic tests for H. pylori include a) serologic testing to detect anti-H. pylori antibodies in blood, b) upper gastrointestinal endoscopy with mucosal biopsies, c) H. pylori culture, including antimicrobial susceptibility testing, and d) detection of H. pylori antigens in stool. Serologic testing, however, cannot distinguish current from old infection. Upper gastrointestinal endoscopic biopsies are submitted for rapid urease testing or histological examination, and this approach has the drawbacks of the invasive nature of endoscopy and the suboptimal performance of histopathology. H. Pylori culture is invasive and cumbersome and detection of H. pylori antigens in stool is limited by the low acceptance of stool testing and suboptimal specificity/sensitivity.

Several diagnostic tests are based on the ability of H. pylori to convert urea to CO₂ and NH₃ using its enzyme urease. H. Pylori produces large amounts of urease which often comprises about five percent of its total protein. Urease activity is assessed in two general ways: Biopsy-based rapid urease testing and various urea breath tests. Biopsy-based rapid urease tests require endoscopy for sample acquisition. Biopsy samples are placed in an agar gel or paper strip containing a pH indicator. In addition to requiring an invasive endoscopy, biopsy-based rapid urease tests provide a less-than-optimal test due to the time required for the diagnosis, which is 3-24 hours, a less than 90% specificity, and reduced sensitivity in children. Moreover, conventional devices, particularly point-of-care devices, are expensive, particularly to assess H. pylori, which discontinuously colonizes the gastroduodenal mucosa.

Conventional testing is performed utilizing instrumentation that ranges from variations of mass spectrometers to IR detectors that are costly and require a trained operator. Breath sample transportation is also an issue with most conventional devices. The limited availability of instruments operable by patients and available at the point of care requires samples to be shipped to central testing facilities, adding cost and inconvenience. In regard to Urea Breath Testing (UBT) for the diagnosis of H. pylori infection, there are two versions of the UBT, based upon the type of urea being used as a substrate: ¹³C labeled urea and ¹⁴C labeled urea. ¹⁴C is a radioactive isotope of carbon. ¹³C is a stable, non-radioactive isotope, encountered in nature. The FDA has approved both ¹³C and ¹⁴C-based UBTs for the diagnosis of H. pylori, though the ¹⁴C-based assay is rarely used. The ¹⁴C-based UBT is associated with exposure to radioactivity, which albeit small for an otherwise healthy adult, is nevertheless present and patients should not be exposed to it in the absence of safe alternatives. It is because of the risk associated with the radioactivity of the ¹⁴C-urea that the ¹⁴C-based UBT is contraindicated in pregnant women and children. See, Using Breath Tests Wisely in a Gastroenterology Practice: An Evidence-Based Review of Indications and Pitfalls in Interpretation, Romagnuolo, et al., Am J Gastroenterology 97:1113-1126 (2002).

A further difficulty arising with the UBT is the high cost of ¹³C-urea, as well as the cost and operational expenses of instruments to detect exhaled ¹³CO₂.

To solve this shortcoming, the present invention departs from detection of ¹³CO₂ by using unlabeled urea as a substrate, detecting ammonia in breath instead of CO₂. The present invention provides an ammonia-specific nanosensor and provides a simple, inexpensive hand-held device for the detection of breath NH₃.

Accordingly, the present invention provides a highly accurate, economical, easy to operate, portable and sufficiently sensitive medical device for diagnosis of H. Pylori. The present invention departs from detection of ¹³CO₂ and provides a simplified assay that uses lower cost unlabeled urea as a substrate.

SUMMARY OF THE INVENTION

The present invention substantially solves the above shortcomings and provides at least the following advantages.

The present invention obviates the need for serologic testing, for upper gastrointestinal endoscopy with mucosal biopsies for the detection of H. pylori, which is invasive and cumbersome, and for detection of H. pylori antigens in stool.

In a preferred embodiment, a medical device is provided to sample breath emitted from a patient's mouth, to analyze ammonia content and the composition of a gaseous sample via contact with sensing electrodes, particularly gold substrates arranged on a TO8 substrate.

Another embodiment of the present invention provides a method for using the medical device of the present invention to analyze a patient's breath sample to diagnose the presence of a medical condition, by obtaining a breath sample from a patient; analyzing volatile components of the patient sample to provide a breath profile that includes both qualitative and quantitative data; comparing the patient's breath profile to a database of breath profiles, with each database profile being characteristic of at least one medical condition, to provide information pertinent to diagnosis of the presence or absence of a medical condition.

In a preferred embodiment, a single sample is used for an independent or multiple tests, which may be combined to produce a template or pattern representative of a patient's condition or representative of the presence of a particular compound or set of compounds. In a preferred embodiment high sensitivity nanomorphs of metal oxides prepared by sol-gel practices are used for a more selective and quantitatively precise analysis.

In a preferred embodiment, the invention utilizes arrays of biocomposite and bio-doped films to provide a low cost, portable analyzer for detection of chemical products of biochemical reactions, such as ammonia and NO, in a real-time manner.

DETAILED DESCRIPTION OF THE FIGURES

The above and other objects, features and advantages of certain exemplary embodiments of the present invention will be more apparent from the following detailed description taken in conjunction with the accompanying drawings, in which:

FIG. 1 is a schematic representation of an embodiment of a test device for the present invention;

FIGS. 2 a and 2 b show heater and sensing electrodes utilized in FIG. 1;

FIGS. 3 a and 3 b show sensor response of the test device of FIG. 1;

FIGS. 4 a and 4 b show NH₃ sensing and sensor response when exposed only to CO₂;

FIG. 5 shows NH₃ sensing with a CO₂ filter; and

FIG. 6 provides a block diagram of an apparatus of an embodiment of the present invention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The below description of detailed construction of preferred embodiments provides a comprehensive understanding of exemplary embodiments of the invention. Accordingly, those of ordinary skill in the art will recognize that various changes and modifications of the embodiments described herein can be made without departing from the scope and spirit of the invention. Descriptions of well-known functions and constructions are omitted for clarity and conciseness.

Analysis of breath and skin emission samples for diagnostic purposes has the advantage that the sample to be analyzed is collected from the patient in a non-invasive manner with a minimum of discomfort or inconvenience. Basic components of the medical device used for analysis in accordance with a preferred embodiment of the present invention are shown in FIG. 1. In preferred embodiments of the invention, breath samples are quantitatively and qualitatively processed. Notably, the sensor is tuned to detect NH₃ levels lower than 50 ppb (<50 ppb) and as high as 500 ppm, thereby covering all NH₃ levels encountered in humans, and in particular in patients undergoing UBT. Quantitative analyzers preferably include a sensing substrate surrounded by a gold substrate surrounded by a TO8 substrate. The medical device of the present invention is preferably qualitatively used to test exhaled gas. Qualitative tests performed by the test device usable with the present invention may test carbon dioxide content, alcohol content, lipid degradation products, aromatic compounds, thio compounds, ammonia and amines or halogenated compounds.

In a preferred embodiment, multiple different tests performed on a single sample may be independent, or may be the result of several tests combined to produce a template or pattern representative of a patient's condition or representative of the presence of a particular compound or set of compounds. The high sensitivity of the nanomorphs of metal oxides prepared by sol-gel practices used in the medical device of the present invention are both more selective and more quantitatively precise than similar information obtained by currently available electronic nose technology. As a result, correlating the data pattern or changes in the data pattern over time identifies a wider range of conditions or compounds.

The present invention departs from detection of ¹³CO₂ and provides a simplified assay that uses unlabeled urea as a substrate and detects ammonia in breath instead of CO₂ utilizing Equation (1):

CO(NH₂)₂+HOH-urease→CO₂+2NH₃  (1)

In an embodiment of the present invention, a nanosensor is provided to detect breath ammonia and a simple, portable, inexpensive hand-held device is thereby provided to detect breath NH₃. The nanosensor is tuned according to the method described below for other breath gases, and the nanosensor is in a preferred embodiment provided as a plug-in component. The sensor is constructed of a metal oxide that is not crystalline, raising sensitivity to ammonia and other gases.

In FIG. 1, a gas sample, i.e. breath or skin emission, accesses analyzer 110 via entry and exit orifices 102 and 104. A stainless steel chamber preferably connects the orifices to avoid absorption/distortion. Sensing electrode 122 and heater electrode 124 are positioned within the analyzer 110. The sensing electrode 122 includes a sensor 130 having gold substrate 132, sensing substrate 134 and TO8 substrate 136. Heater and sensing electrodes 122 and 124 of an embodiment of the present invention are shown in FIGS. 2 a and 2 b. Those of skill in the art recognize use of the TO8 substrate. Hirata et al. in U.S. Pat. No. 5,252,292, the contents of which are incorporated by reference herein, disclose a type of ammonia sensor.

In the present invention, the sensing electrode 124 is selectively tuned by spin or drop coating of sensing substrates generating a film of MoO₃. In a preferred embodiment, a gel-sol synthesis was employed to produce three-dimensional (3-D) networks of nanoparticles, with the sol-gel processing preparing a sol, gelating the sol and removing the solvent. Molybdenum trioxide (MoO3) was prepared by an alkoxide reaction with alcohol according to Equation (2):

Molydenum(VI)Isopropoxide+1-Butanol.fwdarw.Precursor(0.1M)  (2)

The prepared sol was spin coated and drop coated onto sensing substrates generating thin films of MoO₃. The sensing substrates (3 mm×3 mm) were made of Al₂O₃ and were patterned with interdigitated Pt electrodes. Pt heater electrodes were embedded on the rear of the sensor. The amorphous films were then calcined at higher temperatures generating polymorphic form. Differential scanning calorimetry confirmed the phase transformation.

FIG. 3 a shows sensor response to NH₃, with the sensor generating a clear and measurable response to two NH₃ concentrations, 50 and 100 ppb. The measured amounts of ppb, i.e. parts per billion, are much lower than amounts typically expected in human breath, allowing for more accurate and expedited measurement and results. FIG. 3 b shows sensor response to various breath gases, and the specificity regarding same. Shown in FIG. 3 b are NH₃, NO₂, NO, C₃H₆ and NH₃ gases that potentially interfere with NH₃ determination.

FIG. 4 a shows NH₃ sensing without a CO₂ filter, as gas-sensing properties of the nanosensor. As shown in FIGS. 4 a-b, when the sensor was exposed to various concentrations of NH₃ gas in a background mixture of N₂ and O₂ simulating ambient air, NH₃ was detected easily, down to 50 ppb, and even lower concentrations.

In FIG. 4 a, CO₂ and NH₃, each at 1 ppm, produce similar responses to gas pulses, shown as vertical lines in FIG. 4 a. Sensor response when exposed only to CO₂ gas, in the presence of the CO₂ filter, is shown in FIG. 4 b. The CO₂ filter completely eliminates CO₂ from the gas stream, abrogating the sensor response to it.

Sensor specificity, in regard to sensing of NH₃, was evaluated by exposing the sensor to various gases typically encountered in human breath, including NO₂, NO, C₃H₆, and H₂, each up to 490 ppm. Conductivity changes were measured in dry N₂ with 10% O₂. At 440° C. the film was very sensitive to NH₃, with 490 ppm increasing the conductivity by approximately a factor of 70, approximately 17 times greater than the response to the other gases. The NH₃ response, however, was relatively unaffected by 100 ppm of NO₂, NO, C₃H₆, and H₂. X-ray photoelectron spectroscopy (XPS) showed that the increased conductivity in the presence of NH₃ was accompanied by a partial reduction of the surface MoO₃. The resistance of the films increased after extended time at elevated temperatures.

CO₂ is an important component of human breath, with its concentration in expired breath reaching up to 5%. Under test conditions, CO₂ interfered with NH₃ sensing. To overcome this limitation, a commercially available CO₂ filter (NaOH premixed with Vermiculite (V-lite) used in a 10:1 ratio; Decarbite absorption tube, PW Perkins and Co) was used. Decarbite reacts only with highly acidic gases such as CO₂, H₂S, thus excluding the possibility of cross adsorption; and the latter was verified. Exposing the sensor to various concentrations of NH₃ and CO₂, in the presence of N₂ and O₂, indicated that the presence of CO₂ did not affect NH₃ sensing. This was found to be true even when the two gases were at equal concentrations ranging between 0.5 and 10 ppm.

The data shown in FIGS. 4 a-b are from experiments with a low CO₂ concentration (1 ppm). In the present invention, the NaOH Decarbite traps CO₂ more efficiently at high CO₂ concentrations.

FIG. 5 shows NH₃ sensing with a CO₂ filter. In FIG. 5, the sensor is exposed to NH₃ in the presence of the filter, with no interference of the measurement. FIG. 6 shows a prototype for sensing breath, having a sensor, acquisition module, memory/computation module and displays.

Combining NH₃ and CO₂ generated similar results, with the filter eliminating the experimental 1 ppm of CO₂ in the gas stream. Even at low concentrations, interference by CO₂ is eliminated. Operation of the apparatus of the present invention is preferably based on sensor response modifying electrical resistance. That is, the MoO₃ sensor is prepared with properties required for its intended use, with lower limits of detection for NH₃ well below the NH₃ concentrations typically found in human breath and, of course, below the increased NH₃ levels of a positive UBT.

Another embodiment includes colloidal synthesis of hexagonal WO₃ nanowire and sheets. Lithium ion batteries are vital for advancing the field of portable electronics. They operate by reversibly inserting Li+ ions from the electrolyte into the electrodes and for generating electricity. Reversible intercalation of Li+ ions into the host matrix is crucial for battery operation and can be accomplished by having electrode materials that have relatively open crystal structures. Thermodynamically stable crystal structures are typically close-packed, to whereas metastable oxide phases have open lattices that promote very high diffusion rates for intercalating ions.

Building smaller and more efficient batteries is imperative for advancing nanoelectronics. The synthesis of novel materials with reduced dimensionalities for battery electrodes is the key factor in improving battery performance. One-dimensional nanomaterials with high aspect ratio such as nanowires are used to construct miniaturized power units and to increase the surface area of the electrodes in order to increase energy density. Building 3-dimensional architectures of micro-/nano electrodes allows for a reduced footprint area for the battery, while at the same time the high aspect ratio of the nanowires serves to increase the energy density tremendously.

In the area of resistive gas sensing, the attraction of hexagonal WO₃ lies in the structural similarity it shares with the orthorhombic form of Mo0₃. Both these crystal structures have layered oxygen octahedra, in other words an open lattice structure, that provides long paths for small, diffusing gas molecules and facilitates easy removal of oxygen ions from the lattice. The present invention utilizes the key role played by the crystal structure in determining selectivity of the sensing matrix. Orthorhombic MoO₃ has been shown to be selective to ammonia in the presence of other gases. MoO₃ with its low sublimation temperature is not a suitable candidate for prolonged use at elevated temperatures. WO₃ on the other hand has higher structural integrity than MoO₃ and hence ideal for high temperature sensor applications. Also the high aspect ratio of the nanowires will serve to improve the energy density of the batteries without increasing the effective volume of the battery.

Also, metal oxides with lower dimensionalities have been the focus of intense research activity for applications requiring high surface-area to volume ratio such as gas sensing.

Nanowires of h-WO₃ are fabricated by hydrolysis and subsequent annealing of a sub-stoichiometric metal alkoxide precursor (tungsten (V) isopropoxide-W (iPr)5) in air at a maximum temperature limit of 515° C. The sol-gel reaction occurring with the metal alkoxide precursor is outlined in Equation (3):

W(iPr)5+H—OH.fwdarw.W(OH)5+R—OH(1)  (3)

The hydrolysis and subsequent condensation occur by alcoxolation, i.e. by removal of water. The isopropoxide functional group is removed as isopropanol, which then dries out. The precursor for the hexagonal lattice requires an additive for stabilizing the framework. The substoichiometric isopropoxide precursor on reaction with atmospheric moisture results in the formation of H0.24WO₃ that is known to transform to h-WO₃ on oxidation in air. The metal alkoxide is sub-stoichiometric and removal of two molecules of water from the W(OH)5 in Equation (3) results in a lone hydrogen atom that can be accommodated in the interstitial spaces of WO₃ framework.

Advantages of this embodiment include single step synthesis of a novel metastable phase of WO₃; unique crystal structure of the material enables reversible intercalation of Li ions for rechargeable batteries; higher thermal stability; high aspect ratio nanostructures such as nanowires, nanocubes and nanosheets for high surface area to volume ratio applications.

While this invention has been particularly shown and described with reference to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims. 

What is claimed is:
 1. A non-invasive apparatus for diagnosing whether a patient suffers from helicobacter pylori infection, comprising: a portable, hand-held, instrument, including means for sampling the expelled breath of a patient to whom unlabeled urea has been administered, a detector capable of detecting less than 50 parts per billion of unlabeled ammonia in the sample of the said patient's expelled breath and means for displaying and recording data representing the level of ammonia detected.
 2. An apparatus as recited in claim 1 further comprising means for comparing the data representing the level of ammonia detected with a database of information to assist in making said diagnosis.
 3. A non-invasive method for diagnosing whether a patient suffers from helicobacter pylori infection comprising the steps of: administering unlabeled urea to a patient; sampling the patient's expelled breath; passing said sample of expelled breath over a detector capable of detecting less than 50 parts per billion of unlabeled ammonia in the sample of the said patent's expelled breath; displaying and recording data representing the level of ammonia detected.
 4. A method as recited in claim 3 wherein said recorded data is compared with data in a database to make the desired diagnosis. 